Anti-KDM3A / JHDM2A antibody [EPR18875]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling KDM3A / JHDM2A with ab191389 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Weak nuclear staining on cancer cells of human lung cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling KDM3A / JHDM2A with ab191389 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Weak nuclear staining on cancer cells of human bladder cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling KDM3A / JHDM2A with ab191389 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on spermatogenic cells of human testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

KDM3A / JHDM2A was immunoprecipitated from 1mg of Ramos (Human Burkitt's lymphoma cell line) whole cell lysate with ab191389 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab191389 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Ramos whole cell lysate 10μg (Input).

Lane 2 : ab191389 IP in Ramos whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab191389 in Ramos whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-KDM3A / JHDM2A antibody [EPR18875] (ab191389)

Predicted band size: 147 kDa

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• WB

Supplier Data

Western blot - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM3A / JHDM2A antibody [EPR18875] (ab191389) at 1/1000 dilution

All lanes:

Ramos (Human Burkitt's lymphoma cell line) whole cell lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 147 kDa

Observed band size: 147 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

Lane 1 : Wild type HAP1 whole cell lysate (40 μg)
Lane 2 : KDM3A knockout HAP1 whole cell lysate (40 μg)
Lane 3 : HeLa whole cell lysate (40 μg)
Lane 4 : Hek293 whole cell lysate (40 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab191389 observed at 150 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab191389 was shown to specifically react with KDM3A when KDM3A knockout samples were used. Wild-type and KDM3A knockout samples were subjected to SDS-PAGE. ab191389 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 500 dilution and 1/10000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-KDM3A / JHDM2A antibody [EPR18875] (ab191389)

Predicted band size: 147 kDa

false

• WB

Supplier Data

Western blot - Anti-KDM3A / JHDM2A antibody [EPR18875] (AB191389)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 15 seconds; Lane 3 and 4 : 30 seconds; Lane 5 and 6 : 5 seconds.

All lanes:

Western blot - Anti-KDM3A / JHDM2A antibody [EPR18875] (ab191389) at 1/1000 dilution

Lane 1:

Untreated HEK-293 (Human epithelial cell line from embryonic kidney), whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) treated with 100 μM CoCl2 for 24 hours, whole cell lysate at 20 µg

Lane 3:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma), whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 100 μM CoCl2 for 24 hours, whole cell lysate at 20 µg

Lane 5:

Untreated Jurkat (Human T cell leukemia cell line from peripheral blood), whole cell lysate at 20 µg

Lane 6:

Jurkat (Human T cell leukemia cell line from peripheral blood) treated with 100 μM CoCl2 for 24 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 147 kDa,78 kDa

Observed band size: 100 kDa,147 kDa,75 kDa

false